Analysis of high-resolution chromosomes obtained from methotrexate (MTX) synchronized cells of lymphomatous tissue may become an essential tool, along with hisTopathology and immunologic cell markers, in the subclassification of non-Hodgkin's lymphoma (NHL). Using this technique, we have routinely obtained mitoses at the 400 to 850 band stages and have shown that most NHL patients can be successfully analyzed (124 of 128 = 97 percent), have an abnormal karyotype (95%), and 64 percent of them have a specific defect that identifies distinct NHL subgroups. Despite this high degree of specificity, however, we have also found that each of the non-Burkitt NHL subtypes represents a heterogeneous group of diseases and that a specific chromosome defect may be shared among related disorders. Such high degree of complexity may explain previous difficulties in predicting whether NHL patients with a given histopathology and immunologic markers will have a relatively long or short survival. Our basic MTX technique for lymphomatous tissue has now been further improved with the use of bromodeoxycitidine and ethidium bromide to yield routinely 850 to 2000 chromosome bands per haploid set. With the new technique we will study, prospectively, approximately 500 consecutive new NHL patients to correlate chromosomal data with response to new unified treatment protocols from the Eastern Cooperative Oncology Group. Our large-scale study of consecutive patients should provide, for the first time, definitive clues to the clinical course, response to treatment, and survival patterns of individuals with specific primary and secondary chromosomal defects in NHL. It may also establish high-resolution chromosome analysis as a critically important tool in the diagnosis, classification, prognosis, and therapeutic assessment of this complex group of disorders.